The major barrier to the success of gene therapy in the clinic is the lack of safe and efficient DNA delivery methods. Over the past four years we have been developing non-viral delivery systems through synthesis and characterization of DNA delivery polymers such as modified poly(ethylene imines), poly(beta-amino esters), and polylysine graft imidizole acetic acids. Despite progress by us and others, existing non-viral delivery systems are far less efficient than viral vectors. Furthermore, the relationship between the chemical properties and biological function of non-viral delivery systems remains poorly understood. These issues have limited both the use and rational design of effective agents. Therefore, our goal is to develop novel synthetic approaches to and perform an extensive, quantitative study of polymer properties as they relate to gene delivery. Our Specific Aims are: 1) To examine structure/function relationships in polymeric gene delivery through synthesis and characterization of a large library of high transfecting, poly(beta-amino ester)s. Using high throughput methods developed in preliminary studies, a library of 1000's of poly(beta-amino ester)s will be synthesized. This library will be studied, with the goals of 1) identifying highly effective gene delivery polymers and 2) identifying structure/function relationships to guide future polymer development. 2) To develop methods for increasing serum stability and adding celi-specific targeting to DNA delivery polymers. Chemical modifications necessary to add serum resistance to poly(beta-amino ester)s and polyethylenimines will be investigated. Optimal methods for incorporation of the model targeting ligands, galactose, folate will be studied. High throughput methods will be used to formulate Polymer-DNA nanoparticles from blends of different modified and unmodified polymers. These will be tested for DNA delivery efficacy, serum stability, cell-specificity and complement activation using high throughput, cell-based screening. 3) To assay polymeric safety and gene delivery efficacy in mice in vivo. Our best performing polymer-DNA formulations, identified in aims one and two, wilt be assayed in vivo in mice for 1) delivery efficiency, 2) specificity and biodistribution, and 3) complement activation and initial safety profile.